Synthesis, deprotection, analysis and purification of RNA and ribozymes

ABSTRACT

Method for purification and synthesis of RNA molecules and enzymatic RNA molecules in enzymatically active form.

This application is a division of Usman et al., U.S. Ser. No.08/380,734, filed Jan. 30, 1995, (hereby incorporated by referenceherein in totality, including drawings) which is a continuation-in-partof two applications by Usman et al., both entitled "Synthesis,deprotection, analysis and purification of RNA and ribozymes" and filedon Nov. 28, 1994 as U.S. Ser. No. 08/345,516, now abandoned, and on May18, 1994, as U.S. Ser. No. 08/245,736 now abandoned, which is acontinuation-in-part of Dudycz et al. entitled "Preparation of purifiedribozymes in sodium, potassium or magnesium salt form", filed Dec. 14,1993, U.S. Ser. No. 08/167,586 (abandoned), which is a continuation ofDudycz et al. entitled "Preparation of purified ribozymes in sodium,potassium or magnesium salt form", filed May 14, 1992, U.S. Ser. No.07/884,436 (abandoned). All of these prior applications are herebyincorporated by reference herein (including drawings).

BACKGROUND OF THE INVENTION

This invention relates to the synthesis, deprotection, and purificationof enzymatic RNA or modified enzymatic RNA molecules in milligram tokilogram quantities with high biological activity.

The following is a brief history of the discovery and activity ofenzymatic RNA molecules or ribozymes. This history is not meant to becomplete but is provided only for understanding of the invention thatfollows. This summary is not an admission that all of the work describedbelow is prior art to the claimed invention.

Prior to the 1970s it was thought that all genes were direct linearrepresentations of the proteins that they encoded. This simplistic viewimplied that all genes were like ticker tape messages, with each tripletof DNA "letters" representing one protein "word" in the translation.Protein synthesis occurred by first transcribing a gene from DNA intoRNA (letter for letter) and then translating the RNA into protein (threeletters at a time). In the mid 1970s it was discovered that some geneswere not exact, linear representations of the proteins that they encode.These genes were found to contain interruptions in the coding sequencewhich were removed from, or "spliced out" of, the RNA before it becametranslated into protein. These interruptions in the coding sequence weregiven the name of intervening sequences (or introns) and the process ofremoving them from the RNA was termed splicing. After the discovery ofintrons, two questions immediately arose: i) why are introns present ingenes in the first place, and ii) how do they get removed from the RNAprior to protein synthesis? The first question is still being debated,with no clear answer yet available. The second question, how introns getremoved from the RNA, is much better understood after a decade and ahalf of intense research on this question. At least three differentmechanisms have been discovered for removing introns from RNA. Two ofthese splicing mechanisms involve the binding of multiple proteinfactors which then act to correctly cut and join the RNA. A thirdmechanism involves cutting and joining of the RNA by the intron itself,in what was the first discovery of catalytic RNA molecules.

Cech and colleagues were trying to understand how RNA splicing wasaccomplished in a single-celled pond organism called Tetrahymenathermophila. They had chosen Tetrahymena thermophila as a matter ofconvenience, since each individual cell contains over 10,000 copies ofone intron-containing gene (the gene for ribosomal RNA). They reasonedthat such a large number of intron-containing RNA molecules wouldrequire a large amount of (protein) splicing factors to get the intronsremoved quickly. Their goal was to purify these hypothesized splicingfactors and to demonstrate that the purified factors could splice theintron-containing RNA in vitro. Cech rapidly succeeded in getting RNAsplicing to work in vitro, but something unusual was going on. Asexpected, splicing occurred when the intron-containing RNA was mixedwith protein-containing extracts from Tetrahymena, but splicing alsooccurred when the protein extracts were left out. Cech proved that theintervening sequence RNA was acting as its own splicing factor to snipitself out of the surrounding RNA. They published this startlingdiscovery in 1982. Continuing studies in the early 1980's served toelucidate the complicated structure of the Tetrahymena intron and todecipher the mechanism by which self-splicing occurs. Many researchgroups helped to demonstrate that the specific folding of theTetrahymena intron is critical for bringing together the parts of theRNA that will be cut and spliced. Even after splicing is complete, thereleased intron maintains its catalytic structure. As a consequence, thereleased intron is capable of carrying out additional cleavage andsplicing reactions on itself (to form intron circles). By 1986, Cech wasable to show that a shortened form of the Tetrahymena intron could carryout a variety of cutting and joining reactions on tither pieces of RNA.The demonstration proved that the Tetrahymena intron can act as a trueenzyme: i) each intron molecule was able to cut many substrate moleculeswhile the intron molecule remained unchanged, and ii) reactions werespecific for RNA molecules that contained a unique sequence (CUCU) whichallowed the intron to recognize and bind the RNA. Zaug and Cech coinedthe term "ribozyme" to describe any ribonucleic acid molecule that hasenzyme-like properties. Also in 1986, Cech showed that the RNA substratesequence recognized by the Tetrahymena ribozyme could be changed byaltering a sequence within the ribozyme itself. This property has led tothe development of a number of site-specific ribozymes that have beenindividually designed to cleave at other RNA sequences. The Tetrahymenaintron is the most well-studied of what is now recognized as a largeclass of introns, Group I introns. The overall folded structure,including several sequence elements, is conserved among the Group Iintrons, as is the general mechanism of splicing. Like the Tetrahymenaintron, some members of this class are catalytic, i.e. the intron itselfis capable of the self-splicing reaction. Other Group I introns requireadditional (protein) factors, presumably to help the intron fold intoand/or maintain its active structure. While the Tetrahymena intron isrelatively large, (413 nucleotides) a shortened form of at least oneother catalytic intron (SunY intron of phage T4, 180 nucleotides) mayprove advantageous not only because of its smaller size but because itundergoes self-splicing at an even faster rate than the Tetrahymenaintron.

Ribonuclease P (RNAseP) is an enzyme comprised of both RNA and proteincomponents which are responsible for converting precursor tRNA moleculesinto their final form by trimming extra RNA off one of their ends.RNAseP activity has been found in all organisms tested, but thebacterial enzymes have been the most studied. The function of RNAseP hasbeen studied since the mid-1970s by many labs. In the late 1970s, SidneyAltman and his colleagues showed that the RNA component of RNAseP isessential for its processing activity; however, they also showed thatthe protein component also was required for processing under theirexperimental conditions. After Cech's discovery of self-splicing by theTetrahymena intron, the requirement for both protein and RNA componentsin RNAseP was reexamined. In 1983, Altman and Pace showed that the RNAwas the enzymatic component of the RNAseP complex. This demonstratedthat an RNA molecule was capable of acting as a true enzyme, processingnumerous tRNA molecules without itself undergoing any change. The foldedstructure of RNAseP RNA has been determined, and while the sequence isnot strictly conserved between RNAs from different organisms, thishigher order structure is. It is thought that the protein component ofthe RNAseP complex may serve to stabilize the folded RNA in vivo Atleast one RNA position important both to substrate recognition and todetermination of the cleavage site has been identified, however littleelse is known about the active site. Because tRNA sequence recognitionis minimal, it is clear that some aspect(s) of the tRNA structure mustalso be involved in substrate recognition and cleavage activity. Thesize of RNAseP RNA (>350 nucleotides), and the complexity of thesubstrate recognition, may limit the potential for the use of anRNAseP-like RNA in therapeutics. However, the size of RNAseP is beingtrimmed down (a molecule of only 290 nucleotides functions reasonablywell). In addition, substrate recognition has been simplified by therecent discovery that RNAseP RNA can cleave small RNAs lacking thenatural tRNA secondary structure if an additional RNA (containing a"guide" sequence and a sequence element naturally present at the end ofall tRNAs) is present as well.

Symons and colleagues identified two examples of a self-cleaving RNAthat differed from other forms of catalytic RNA already reported. Symonswas studying the propagation of the avocado sunblotch viroid (ASV), anRNA virus that infects avocado plants. Symons demonstrated that aslittle as 55 nucleotides of the ASV RNA was capable of folding in such away as to cut itself into two pieces. It is thought that in vivoself-cleavage of these RNAs is responsible for cutting the RNA intosingle genome-length pieces during viral propagation. Symons discoveredthat variations on the minimal catalytic sequence from ASV could befound in a number of other plant pathogenic RNAs as well. Comparison ofthese sequences revealed a common structural design consisting of threestems and loops connected by central loop containing many conserved(invariant from one RNA to the next) nucleotides. The predictedsecondary structure for this catalytic RNA reminded the researchers ofthe head of a hammer consisting of three double helical domains, stemsI, II and III and a catalytic core (FIG. 1 and 2a); thus it was named assuch. Uhlenbeck was successful in separating the catalytic region of theribozyme from that of the substrate. Thus, it became possible toassemble a hammerhead ribozyme from 2 (or 3) small synthetic RNAs. A19-nucleotide catalytic region and a 24-nucleotide substrate,representing division of the hammerhead domain, along the axes of stemsI and II (FIG. 2b) were sufficient to support specific cleavage. Thecatalytic domain of numerous hammerhead ribozymes have now been studiedby both the Uhlenbeck and Symons groups with regard to defining thenucleotides required for specific assembly and catalytic activity anddetermining the rates of cleavage under various conditions.

Haseloff and Gerlach showed it was possible to divide the domains of thehammerhead ribozyme in a different manner, division of the hammerheaddomain along the axes of stems I and III (FIG. 2c). By doing so, theyplaced most of the required sequences in the strand that didn't get cut(the ribozyme) and only a required UH where H=C, A, U in the strand thatdid get cut (the substrate). This resulted in a catalytic ribozyme thatcould be designed to cleave any UH RNA sequence embedded within a longer"substrate recognition" sequence. The specific cleavage of a long mRNA,in a predictable manner using several such hammerhead ribozymes, wasreported in 1988. A further development was the division of thecatalytic hammerhead domain along the axes of stems III and II (FIG. 2d,Jeffries and Symons, Nucleic Acids. Res. 1989, 17, 1371-1377.)

One plant pathogen RNA (from the negative strand of the tobacco ringspotvirus) undergoes self-cleavage but cannot be folded into the consensushammerhead structure described above. Bruening and colleagues haveindependently identified a 50-nucleotide catalytic domain for this RNA.In 1990, Hampel and Tritz succeeded in dividing the catalytic domaininto two parts that could act as substrate and ribozyme in amultiple-turnover, cutting reaction (FIG. 3). As with the hammerheadribozyme, the hairpin catalytic portion contains most of the sequencesrequired for catalytic activity while only a short sequence (GUC in thiscase) is required in the target. Hampel and Tritz described the foldedstructure of this RNA as consisting of a single hairpin and coined theterm "hairpin" ribozyme (Bruening and colleagues use the term"paperclip" for this ribozyme motif, see, FIG. 3). Continuingexperiments suggest an increasing number of similarities between thehairpin and hammerhead ribozymes in respect to both binding of targetRNA and mechanism of cleavage. At the same time, the minimal size of thehairpin ribozyme is still 50-60% larger than the minimal hammerheadribozyme.

Hepatitis Delta Virus (HDV) is a virus whose genome consists ofsingle-stranded RNA. A small region (˜80 nucleotides, FIG. 4) in boththe genomic RNA, and in the complementary anti-genomic RNA, issufficient to support self-cleavage. As the most recently discoveredribozyme, HDV's ability to self-cleave has only been studied for a fewyears, but is interesting because of its connection to a human disease.In 1991, Been and Perrotta proposed a secondary structure for the HDVRNAs that is conserved between the genomic and anti-genomic RNAs and isnecessary for catalytic activity. Separation of the HDV RNA into"ribozyme" and "substrate" portions has recently been achieved by Been,but the rules for targeting different substrate RNAs have not yet beendetermined fully (see, FIG. 4). Been has also succeeded in reducing thesize of the HDV ribozyme to ˜60 nucleotides.

The Table I lists some of the characteristics of the ribozymes discussedabove.

Ribozymes are RNA molecules having an enzymatic activity which is ableto repeatedly cleave other separate RNA molecules in a nucleotide basesequence specific manner. It is said that such enzymatic RNA moleculescan be targeted to virtually any RNA transcript and efficient cleavagehas been achieved in vitro. Kim et al., 84 Proc. Nat. Acad. of Sci. USA8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA3030, 1988; and Jefferies et al., 17 Nucleic Acid Research 1371, 1989.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseribozymes. In general, enzymatic nucleic acids act by first binding to atarget RNA. Such binding occurs through the target binding portion of aenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets.

By "enzymetic RNA molecule" it is meant an RNA molecule which hascomplementarity in a substrate binding region to a specified mRNAtarget, and also has an enzymetic activity which is active tospecifically cleave that mRNA. That is, the enzymetic RNA molecule isable to intermolecularly cleave mRNA and thereby inactivate a targetmRNA molecule. This complementarity functions to allow sufficienthybridization of the enzymetic RNA molecule to the target RNA to allowthe cleavage to occur. One hundred percent complementarity is preferred,but complementarity as low as 50-75% may also be useful in thisinvention. For in vivo treatment, complementarity between 30 and 45bases is preferred; although lower numbers are also useful.

By "complementary" is meant a nucleotide sequence that can form hydrogenbond(s) with other nucleotide sequence by either traditionalWatson-Crick or other non-traditional types (for example Hoogsteen type)of base-paired interactions.

The enzymetic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (WooIf, T. M., et el., 1992, Proc.Natl. Aced. Sci. USA, 89, 7305-7309). Thus, the specificity of action ofa ribozyme is greater than that of an antisense oligonucieotide bindingthe same RNA site.

In preferred embodiments of this invention, the enzymatic nucleic acidmolecule is formed in a hammerhead or hairpin motif, but may also beformed in the motif of a hepatitis delta virus, group I intron or RNasePRNA (in association with an RNA guide sequence) or Neurospora VS RNA.Examples of such hammerhead motifs are described by Rossi et al., 1992,Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampelet al., EPA 0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, andHampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of thehepatitis delta virus motif is described by Perrotta and Been, 1992Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad, Sci, USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al.,U.S. Pat. No. 4,987,071. These specific motifs are not limiting in theinvention and those skilled in the art will recognize that all that isimportant in an enzymatic nucleic acid molecule of this invention whichis complementary to one or more of the target gene RNA regions, and thatit have nucleotide sequences within or surrounding that substratebinding site which impart an RNA cleaving activity to the molecule.

Enzymatic nucleic acids act by first binding to a target RNA (or DNA,see Cech U.S. Pat. No. 5,180,818). Such binding occurs through thetarget binding portion of an enzymatic nucleic acid which is held inclose proximity to an enzymatic portion of molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Cleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The invention provides a method for producing a class of enzymaticcleaving agents or antisense molecules which exhibit a high degree ofspecificity for the RNA or DNA of a desired target. The enzymaticnucleic acid or antisense molecule is preferably targeted to a highlyconserved sequence region of a target such that specific treatment of adisease or condition can be provided with a single enzymatic nucleicacid. Such, nucleic acid molecules can be delivered exogenously tospecific cells as required. In the preferred hammerhead motif the smallsize (less than 60 nucleotides, preferably between 30-40 nucleotides inlength) of the molecule allows the cost of treatment to be reducedcompared to other ribozyme motifs.

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small enzymatic nucleicacid motifs (e.g., of the hammerhead structure) are used for exogenousdelivery. The simple structure of these molecules increases the abilityof the enzymatic nucleic acid to invade targeted regions of the mRNAstructure. Unlike the situation when the hammerhead structure isincluded within longer transcripts, there are no non-enzymatic nucleicacid flanking sequences to interfere with correct folding of theenzymatic nucleic acid structure or with complementary regions.

Generally, RNA is synthesized and purified by methodologies based on:tetrazole to activate the RNA amidite, NH₄ OH to remove the exocyclicamino protecting groups, tetra-n-butylammonium fluoride (TBAF) to removethe 2'-OH alkylsilyl protecting groups, and gel purification andanalysis of the deprotected RNA. In particular this applies to, but isnot limited to, a certain class of RNA molecules, ribozymes. These maybe formed either chemically or using enzymatic methods. Examples of thechemical synthesis, deprotection, purification and analysis proceduresare provided by Usman et al., 1987 J. American Chem. Soc., 109, 7845,Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341, Perreauit et al.Biochemistry 1991, 30 4020-4025, and Slim and Gait Nucleic Acids Res.1991, 19, 1183-1188. Odai et al. FEBS Lett. 1990, 267, 150-152 describesa reverse phase chromatographic purification of RNA fragments used toform a ribozyme. All the above noted references are all herebyincorporated by reference herein.

The aforementioned chemical synthesis, deprotection, purification andanalysis procedures are time consuming (10-15 m coupling times) and mayalso be affected by inefficient activation of the RNA amidites bytetrazole, time consuming (6-24 h) and incomplete deprotection of theexocyclic amino protecting groups by NH₄ OH, time consuming (6-24 h),incomplete and difficult to desalt TBAF-catalyzed removal of thealkylsilyl protecting groups, time consuming and low capacitypurification of the RNA by gel electrophoresis, and low resolutionanalysis of the RNA by gel electrophoresis.

Imazawa and Eckstein, 1979 J. Org. Chem., 12, 2039, describe thesynthesis of 2'-amino-2'-deoxyribofuranosyl purines. They state that

"To protect the 2'-amino function, we selected the trifluoroacetyl groupwhich can easily be removed."

SUMMARY OF THE INVENTION

This invention concerns the chemical synthesis, deprotection, andpurification of RNA, enzymatic RNA or modified RNA molecules in greaterthan milligram quantities with high biological activity. Applicant hasdetermined that the synthesis of enzymatically active RNA in high yieldand quantity is dependent upon certain critical steps used during itspreparation. Specifically, it is important that the RNA phosphoramiditesare coupled efficiently in terms of both yield and time, that correctexocyclic amino protecting groups be used, that the appropriateconditions for the removal of the exocyclic amino protecting groups andthe alkylsilyl protecting groups on the 2'-hydroxyl are used, and thatthe correct work-up and purification procedure of the resulting ribozymebe used.

To obtain a correct synthesis in terms of yield and biological activityof a large RNA molecule (i.e., about 30 to 40 nucleotide bases), theprotection of the amino functions of the bases requires either amide orsubstituted amide protecting groups, which must be, on the one hand,stable enough to survive the conditions of synthesis, and on the otherhand, removable at the end of the synthesis. These requirements are metby the amide protecting groups shown in FIG. 6, in particular, benzoylfor adenosine, isobutyryl or benzoyl for cytidine, and isobutyryl forguanosine, which may be removed at the end of the synthesis byincubating the RNA in NH₃ /EtOH (ethanolic ammonia) for 20 h at 65° C.In the case of the phenoxyacetyl type protecting groups shown in FIG. 6on guanosine and adenosine and acetyl protecting groups on cytidine, anincubation in ethanolic ammonia for 4 h at 65° C. is used to obtaincomplete removal of these protecting groups. Removal of the alkylsilyl2'-hydroxyl protecting groups can be accomplished using atetrahydrofuran solution of TBAF at room temperature for 8-24 h.

The most quantitative procedure for recovering the fully deprotected RNAmolecule is by either ethanol precipitation, or an anion exchangecartridge desalting, as described in Scaringe et al. Nucleic Acids Res.1990, 18, 5433-5341. The purification of the long RNA sequences may beaccomplished by a two-step chromatographic procedure in which themolecule is first purified on a reverse phase column with either thetrityl group at the 5' position on or off. This purification isaccomplished using an acetonitrile gradient with triethylammonium orbicarbonate salts as the aqueous phase. In the case of the trityl onpurification, the trityl group may be removed by the addition of an acidand drying of the partially purified RNA molecule. The finalpurification is carried out on an anion exchange column, using alkalimetal perchlorate salt gradients to elute the fully purified RNAmolecule as the appropriate metal salts, e.g. Na⁺, Li⁺ etc. A finalde-salting step on a small reverse-phase cartridge completes thepurification procedure. Applicant has found that such a procedure notonly fails to adversely affect activity of a ribozyme, but may improveits activity to cleave target RNA molecules.

Applicant has also determined that significant (see Tables 2-4)improvements in the yield of desired full length product (FLP) can beobtained by:

1. Using 5-S-alkyltetrazole at a delivered or effective concentration of0.25-0.5M or 0.15-0.35M for the activation of the RNA (or analogue)amidite during the coupling step. (By delivered is meant that the actualamount of chemical in the reaction mix is known. This is possible forlarge scale synthesis since the reaction vessel is of size sufficient toallow such manipulations. The term effective means that available amountof chemical actually provided to the reaction mixture that is able toreact with the other reagents present in the mixture. Those skilled inthe art will recognize the meaning of these terms from the examplesprovided herein.) The time for this step is shortened from 10-15 m, videsupra, to 5-10 m. Alkyl, as used herein, refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain, and cyclic alkylgroups. Preferably, the alkyl group has 1 to 12 carbons. More preferablyit is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4carbons. The alkyl group may be substituted or unsubstituted. Whensubstituted the substituted group(s) is preferably, hydroxyl, cyano,alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino, or SH. The term also includesalkenyl groups which are unsaturated hydrocarbon groups containing atleast one carbon-carbon double bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkenyl group has 1to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkenyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂,amino, or SH. The term "alkyl" also includes alkynyl groups which havean unsaturated hydrocarbon group containing at least one carbon-carbontriple bond, including straight-chain, branched-chain, and cyclicgroups. Preferably, the alkynyl group has 1 to 12 carbons. Morepreferably it is a lower alkynyl of from 1 to 7 carbons, more preferably1 to 4 carbons. The alkynyl group may be substituted or unsubstituted.When substituted the substituted group(s) is preferably, hydroxyl,cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An "aryl" group refers to anaromatic group which has at least one ring having a conjugated πelectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An"alkylaryl" group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above. Carbocyclic arylgroups are groups wherein the ring atoms on the aromatic ring are allcarbon atoms. The carbon atoms are optionally substituted. Heterocyclicaryl groups are groups having from 1 to 3 heteroatoms as ring atoms inthe aromatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An"amide" refers to an --C(O)--NH--R, where R is either alkyl, aryl,alkylaryl or hydrogen. An "ester" refers to an --C(O)--OR', where R iseither alkyl, aryl, alkylaryl or hydrogen.

2. Using 5-S-alkyltetrazole at an effective, or final, concentration of0.1-0.35M for the activation of the RNA (or analogue) amidite during thecoupling step. The time for this step is shortened from 10-15m, videsupra,, to 5-10 m.

3. Using alkylamine (MA, where alkyl is preferably methyl, ethyl, propylor butyl) or NH₄ OH/alkylamine (AMA, with the same preferred alkylgroups as noted for MA) @ 65° C. for 10-15 m to remove the exocyclicamino protecting groups (vs 4-20 h @55°-65° C. using NH₄ OH/EtOH or NH₃/EtOH, vide supra). Other alkylamines, e.g. ethylamine, propylamine,butylamine etc. may also be used.

4. Using anhydrous triethylamineohydrogen fluoride (aHF.TEA) @ 65° C.for 0.5-1.5 h to remove the 2'-hydroxyl alkylsilyl protecting group (vs8-24 h using TBAF, vide supra or TEA.3HF for 24 h (Gasparutto et al.Nucleic Acids Res. 1992, 20, 5159-5166). Other alkylamine.HF complexesmay also be used, e.g. trimethylamine or diisopropylethylamine.

5. The use of anion-exchange resins to purify and/or analyze the fullydeprotected RNA. These resins include, but are not limited to,quartenary or tertiary amino derivatized stationary phases such assilica or polystyrene. Specific examples include Dionex-NA100®, Mono-Q®,Poros-Q®.

Thus, in various aspects, the invention features an improved method forthe coupling of RNA phosphoramidites; for the removal of amide orsubstituted amide protecting groups; and for the removal of 2'-hydroxylalkylsilyl protecting groups. Such methods enhance the production of RNAor analogs of the type described above (e.g., with substituted2'-groups), and allow efficient synthesis of large amounts of such RNA.Such RNA may also have enzymatic activity and be purified without lossof that activity. While specific examples are given herein, those in theart will recognize that equivalent chemical reactions can be performedwith the alternative chemicals noted above, which can be optimized andselected by routine experimentation.

In another aspect, the invention features an improved method for thepurification or analysis of RNA or enzymatic RNA molecules (e.g. 28-70nucleotides in length) by passing said RNA or enzymatic RNA moleculeover an HPLC, e.g., reverse phase and/or an anion exchangechromatography column. The method of purification improves the catalyticactivity of enzymatic RNAs over the gel purification method (see FIG.8).

This invention also features a method for preparation of pureenzymatically active ribozymes (of size between 28 and 70 nucleotidebases) in sodium, potassium or magnesium salt form by a two steppurification method. Generally the method is applicable to bothsynthetically and enzymatically produced ribozymes, and entails use ofhigh performance liquid chromatography (HPLC) techniques on reversephase columns. Unlike gel purification, HPLC purification as describedin this application can be applied to virtually unlimited amounts ofpurified material. This allows generation of kilogram quantities ofribozymes in each purification batch.

Thus, in another aspect, the invention features a method forpurification of an enzymatic RNA molecule of 28-70 nucleotide bases bypassing that enzymatic RNA molecule over a high pressure liquidchromatography column. Surprisingly, applicant has determined thatenzymatically active ribozymes can be purified in the desired salt formby the described HPLC or anion exchange methodology.

In preferred embodiments, the method includes passing the enzymaticallyactive RNA molecule over a reverse phase HPLC column; the enzymaticallyactive RNA molecule is produced in a synthetic chemical method and notby an enzymatic process; and the enzymatic RNA molecule contains a5'-DMT group, and the 5'-DMT-containing enzymatically active RNAmolecule is passed over a reverse phase HPLC column to separate it fromother RNA molecules.

In a related aspect, the invention features pure ribozyme in a Na⁺, K⁺,or Mg²⁺ salt form. By "pure" is meant that the ribozyme is preferablyprovided free of other contaminants, and is at least 85% in the desiredsalt form.

Thus, the purification of long RNA molecules may be accomplished usinganion exchange chromatography, particularly in conjunction with alkaliperchlorate salts. This system may be used to purify very long RNAmolecules. In particular, it is advantageous to use a Dionex NucleoPak100© or a Pharmacia Mono Q® anion exchange column for the purificationof RNA by the anion exchange method. This anion exchange purificationmay be used following a reverse-phase purification or prior to reversephase purification. This method results in the formation of a sodiumsalt of the ribozyme during the chromatography. Replacement of thesodium alkali earth salt by other metal salts, e.g., lithium, magnesiumor calcium perchlorate, yields the corresponding salt of the RNAmolecule during the purification.

In the case of the 2-step purification procedure, in which the firststep is a reverse phase purification followed by an anion exchange step,the reverse phase purification is best accomplished using polymeric,e.g. polystyrene based, reverse-phase media, using either a 5'-trityl-onor 5'-trityl-off method. Either molecule may be recovered using thisreverse-phase method, and then, once detritylated, the two fractions maybe pooled and then submitted to an anion exchange purification step asdescribed above.

The method includes passing the enzymatically active RNA molecule over areverse phase HPLC column; the enzymatically active RNA molecule isproduced in a synthetic chemical method and not by an enzymatic process;and the enzymatic RNA molecule is partially blocked, and the partiallyblocked enzymatically active RNA molecule is passed over a reverse phaseHPLC column to separate it from other RNA molecules.

In more preferred embodiments, the enzymatically active RNA molecule,after passage over the reverse phase HPLC column, is deprotected andpassed over a second reverse phase HPLC column (which may be the same asthe reverse phase HPLC column), to remove the enzymatic RNA moleculefrom other components. In addition, the column is a silica or organicpolymer-based C4, C8 or C18 column having a porosity of at least 125 Å,preferably 300 Å, and a particle size of at least 2 μm, preferably 5 μm.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings:

FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain (SEQ ID Nos. 1 and 2) known in the art. Stem II can be ≧2base-pairs long.

FIG. 2a is a diagrammatic representation of the hammerhead ribozymedomain known in the art;

FIG. 2b is a diagrammatic representation of the hammerhead ribozyme asdivided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate andenzyme portion;

FIG. 2c is a similar diagram showing the hammerhead divided by Haseloffand Gerlach (1988, Nature, 334, 585-591) into two portions.

FIG. 2d is a similar diagram showing the hammerhead divided by Jeffriesand Symons (1989, Nucl, Acids. Res., 17, 1371-1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of ahairpin ribozyme (SEQ ID Nos. 3 and 4). Helix 2 (H2) is provided with aleast 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can beoptionally provided of length 2 or more bases (preferably 3-20 bases,i.e., m is from 1-20 or more). Helix 2 and helix 5 may be covalentlylinked by one or more bases (i.e., r is ≧1 base). Helix 1, 4 or 5 mayalso be extended by 2 or more base pairs (e.g., 4-20 base pairs) tostabilize the ribozyme structure, and preferably is a protein bindingsite. In each instance, each N and N' independently is any normal ormodified base and each dash represents a potential base-pairinginteraction. These nucleotides may be modified at the sugar, base orphosphate. Complete base-pairing is not required in the helices, but ispreferred. Helix 1 and 4 can be of any size (i.e., o and p is eachindependently from 0 to any number, e.g., 20) as long as somebase-pairing is maintained. Essential bases are shown as specific basesin the structure, but those in the art will recognize that one or moremay be modified chemically (abasic, base, sugar and/or phosphatemodifications) or replaced with another base without significant effect.Helix 4 can be formed from two separate molecules, i.e., without aconnecting loop. The connecting loop when present may be aribonucleotide with or without modifications to its base, sugar orphosphate. "q" is ≧2 bases. The connecting loop can also be replacedwith a non-nucleotide linker molecule. H, refers to bases A, U or C. Yrefers to pyrimidine bases. "₋₋₋₋₋₋ " refers to a chemical bond.

FIG. 4A is a representation of the general structure of the hepatitisdelta virus ribozyme domain (SEQ ID No. 5) known in the art.

FIG. 4B is a representation of the general structure of theself-cleaving VS RNA ribozyme domain (Seq. ID NO.6).

FIG. 5 is a diagrammatic representation of the solid-phase synthesis ofRNA.

FIG. 6 is a diagrammatic representation of exocyclic amino protectinggroups for nucleic acid synthesis.

FIG. 7 is a diagrammatic representation of the deprotection of RNA.

FIG. 8 is a graphical representation of the cleavage of an RNA substrateby ribozymes synthesized, deprotected and purified using the improvedmethods described herein.

FIGS. 9 and 10 are copies of HPLC results showing purification ofribozyme from failure sequences (FIG. 9) and other contaminants (FIG.10).

FIG. 11 is a schematic representation of a two pot deprotectionprotocol. Base deprotection is carried out with aqueous methyl amine at65° C. for 10 min. The sample is dried in a speed-vac for 2-24 hoursdepending on the scale of RNA synthesis. Silyl protecting group at the2'-hydroxyl position is removed by treating the sample with 1.4Manhydrous HF at 65° C. for 1.5 hours.

FIG. 12 is a schematic representation of a one pot deprotection of RNAsynthesized using RNA phosphoramidite chemistry. Anhydrous methyl amineis used to deprotect bases at 65° C. for 15 min. The sample is allowedto cool for 10 min before adding TEA.3HF reagent, to the same pot, toremove protecting groups at the 2'-hydroxyl position. The deprotectionis carried out for 1.5 hours.

FIG. 13 is a HPLC profile of a 36 nt long ribozyme, targeted to site A.The RNA is deprotected using either the two pot or the one potdeprotection protocol. The peaks corresponding to full-length RNA isindicated.

FIG. 14 is a graph comparing RNA cleavage activity of ribozymesdeprotected by two pot vs one pot deprotection protocols.

FIG. 15 is a schematic representation of an improved method ofsynthesizing RNA containing phosphorothioate linkages.

FIG. 16 shows RNA cleavage reaction catalyzed by ribozymes containingphosphorothioate linkages. Hammerhead ribozyme targeted to site A issynthesized such that 4 nts at the 5' end contain phosphorothioatelinkages. P═O refers to ribozyme without phosphorothioate linkages. P═Srefers to ribozyme with phosphorothioate linkages.

FIG. 17 is a schematic representation of synthesis of2'-N-phtalimido-nucleoside phosphoramidite.

EXAMPLES

The following are non-limiting examples showing the synthesis ofRNA-containing nucleic acids and the testing of the enzymatic activityof these molecules when they are catalytic RNAs.

Activation

The synthesis of RNA molecules may be accomplished chemically orenzymatically. In the case of chemical synthesis the use of tetrazole asan activator of RNA phosphoramidites is known (Usman et al. J. Am. Chem.Soc. 1987, 109, 7845-7854). In this, and subsequent reports, a 0.5Msolution of tetrazole is allowed to react with the RNA phosphoramiditeand couple with the polymer bound 5'-hydroxyl group for 10 m. Applicanthas determined that using 0.25-0.5M solutions of 5-S-alkyltetrazoles foronly 5 min gives equivalent or better results. The following exemplifiesthe procedure.

Example 1 Synthesis of RNA and Ribozymes Using 5-S-Alkyltetrazoles asActivating Agent

The method of synthesis used follows the general procedure for RNAsynthesis as described in Usman et al,, 1987 supra and in Scaringe etal., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5'-end, and phosphoramidites at the 3'-end. The major differenceused, was the activating agent, 5-S-ethyl or -methyltetrazole @ 0.25Mconcentration for 5 min.

All small scale syntheses were conducted on a 394 (ABI) synthesizerusing a modified 2.5 μmol scale protocol with a reduced 5 min couplingstep for alkylsilyl protected RNA and 2.5 m coupling step for2'-O-methylated RNA. A 6.5-fold excess (162.5 μL of 0.1M=32.5 μmol) ofphosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 μL of0.25M=100 μmol) relative to polymer-bound 5'-hydroxyl was used in eachcoupling cycle. Average coupling yields on the 394, determined bycolorimetric quantitation of the trityl fractions, was 97.5-99%. Otheroligonucleotide synthesis reagents for the 394: Detritylation solutionwas 2% TCA in methylene chloride; capping was performed with 16%N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine inTHF; oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water in THF.Fisher Synthesis Grade acetonitrile was used directly from the reagentbottle. S-Ethyl tetrazole solution (0.25M in acetonitrile) was made upfrom the solid obtained from Applied Biosystems.

All large scale syntheses were conducted on a modified (eight amiditeport capacity) 390Z (ABI) synthesizer using a 25 μmol scale protocolwith a 5-15 min coupling step for alkylsilyl protected RNA and 7.5 mcoupling step for 2'-O-methylated RNA. A six-fold excess (1.5mL of0.1M=150 μmol) of phosphoramidite and a forty-five-fold excess ofS-ethyl tetrazole (4.5 mL of 0.25M=1125 μmol) relative to polymer-bound5'-hydroxyl was used in each coupling cycle. Average coupling yields onthe 390Z, determined by colorimetric quantitation of the tritylfractions, was 95.0-96.7%. Oligonucleotide synthesis reagents for the390Z: Detritylation solution was 2% DCA in methylene chloride; cappingwas performed with 16% N-Methyl imidazole in THF and 10% aceticanhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM I₂, 49mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile wasused directly from the reagent bottle. S-Ethyl tetrazole solution(0.25-0.5M in acetonitrile) was made up from the solid obtained fromApplied Biosystems.

Table 1 is a summary of the results obtained using the improvementsoutlined in this application for the large-scale synthesis of RNA andmodified RNAs.

Deprotection

The first step of the deprotection of RNA molecules may be accomplishedby removal of the exocyclic amino protecting groups with either NH₄OH/EtOH:3/1 (Usman et al. J. Am. Chem, Soc. 1987, 109, 7845-7854) or NH₃/EtOH (Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341) for ˜20 h@ 55°-65° C. Applicant has determined that the use of methylamine or NH₄OH/methylamine for 10-15 min @ 55°-65° C. gives equivalent or betterresults. The following exemplifies the procedure.

Example 2 RNA and Ribozyme Deprotection of Exocyclic Amino ProtectingGroups Using Methylamine (MA) or NH₄ OH/Methylamine (AMA)

The polymer-bound oligonucleotide, either trityl-on or off, wassuspended in a solution of methylamine (MA) or NH₄ OH/methylamine (AMA)@ 55°-65° C. for 5-15 min to remove the exocyclic amino protectinggroups. The polymer-bound oligoribonucleotide was transferred from thesynthesis column to a 4 mL glass screw top vial. NH₄ OH and aqueousmethylamine were pre-mixed in equal volumes. 4 mL of the resultingreagent was added to the vial, equilibrated for 5 m at RT and thenheated at 55° or 65° C. for 5-15 min. After cooling to -20° C., thesupernatant was removed from the polymer support. The support was washedwith 1.0 mL of EtOH:MeCN:H₂ O/3:1:1, vortexed and the supernatant wasthen added to the first supernatant. The combined supernatants,containing the oligoribonucleotide, were dried to a white powder. Thesame procedure was followed for the aqueous methylamine reagent.

Table III is a summary of the results obtained using the improvementsoutlined in this application for base deprotection.

The second step of the deprotection of RNA molecules may be accomplishedby removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for8-24 h (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845-7854). Applicanthas determined that the use of anhydrous TEA.HF in N-methylpyrrolidine(NMP) for 0.5-1.5 h @ 55°-65° C. gives equivalent or better results. Thefollowing exemplifies this procedure.

Example 3 RNA and Ribozyme Deprotection of 2'-Hydroxyl AlkylsilylProtecting Groups Using Anhydrous TEA.HF

To remove the alkylsilyl protecting groups, the ammonia-deprotectedoligoribonucleotide was resuspended in 250 μL of 1.4M anhydrous HFsolution (1.5 mL N-methylpyrrolidine, 750 μL TEA and 1.0 mL TEA.3HF) andheated to 65° C. for 1.5 h. 9 mL of 50 mM TEAB was added to quench thereaction. The resulting solution was loaded onto a Qiagen 500® anionexchange cartridge (Qiagen Inc.) prewashed with 10 mL of 50 mM TEAB.After washing the cartridge with 10 mL of 50 mM TEAB, the RNA was elutedwith 10 mL of 2M TEAB and dried down to a white powder.

Table IV is a summary of the results obtained using the improvementsoutlined in this application for alkylsilyl deprotection.

RNA and Ribozyme Purification

The method of this invention generally features HPLC purification ofribozymes. An example of such purification is provided below in which asynthetic ribozyme produced on a solid phase is blocked. This materialis then released from the solid phase by a treatment with methanolicammonia, subsequently treated with tetrabutylammonium fluoride, andpurified on reverse phase HPLC to remove partially blocked ribozyme from"failure" sequences (FIG. 9). Such "failure" sequences are RNA moleculeswhich have a nucleotide base sequence shorter to that of the desiredenzymatic RNA molecule by one or more of the desired bases in a randommanner, and possess free terminal 5'-hydroxyl group. This terminal5'-hydroxyl in a ribozyme with the correct sequence is still blocked bylipophilic dimethoxytrityl group. After such partially blocked enzymaticRNA is purified, it is deblocked by a standard procedure, and passedover the same or a similar HPLC reverse phase column to remove othercontaminating components, such as other RNA molecules or nucleotides orother molecules produced in the deblocking and synthetic procedures(FIG. 10). The resulting molecule is the native enzymatically activeribozyme in a highly purified form.

Below are provided examples of such a method. These examples can bereadily. scaled up to allow production and purification of gram or evenkilogram quantities of ribozymes.

Example 4 HPLC Purification, Reverse-Phase

In this example solid phase phosphoramidite chemistry was employed forsynthesis of a ribozyme. Monomers used were 2'-t-butyl-dimethylsilylcyanoethylphosphoramidites of uridine, N-benzoyl-cytosine,N-phenoxyacetyl adenosine, and guanosine (Glen Research, Sterling, Va.).

Solid phase synthesis was carried out on either an ABI 394 or 380BDNA/RNA synthesizer using the standard protocol provided with eachmachine. The only exception was that the coupling step was increasedfrom 10 to 12 minutes. The phosphoramidite concentration was 0.1M.Synthesis was done on a 1 μmol scale using a 1 μmol RNA reaction column(Glen Research). The average coupling efficiencies were between 97% and98% for the 394 model and between 97% and 99% for the 380B model, asdetermined by a calorimetric measurement of the released trityl cation.The final 5'-DMT group was not removed.

After synthesis, the ribozymes were cleaved from the CPG support, andthe base and phosphotriester moieties were deprotected in a sterile vialby incubation in dry ethanolic ammonia (2 mL) at 55° C. for 16 hours.The reaction mixture was cooled on dry ice, Later, the cold liquid wastransferred into a sterile screw cap vial and lyophilized.

To remove the 2'-t-butyldimethylsilyl groups from the ribozyme theobtained residue was suspended in 1M tetra-n-butylammonium fluoride indry THF (TBAF), using a 20-fold excess of the reagent for every silylgroup, for 16 hours at ambient temperature. The reaction was quenched byadding an equal volume of a sterile 1M triethylamine acetate, pH 6.5.The sample was cooled and concentrated on a SpeedVac to half of theinitial volume.

The ribozymes were purified in two steps by HPLC on a C4 300 Å 5 μmDeltaPak column in an acetonitrile gradient.

The first step, or "trityl on" step, was a separation of5'-DMT-protected ribozyme(s) from failure sequences lacking a 5'-DMTgroup. Solvents used for this step were: A (0.1M triethylammoniumacetate, pH 6.8) and B (acetonitrile). The elution profile was: 20% Bfor 10 minutes, followed by a linear gradient of 20% B to 50% B over 50minutes, 50% B for 10 minutes, a linear gradient of 50% B to 100% B over10 minutes, and a linear gradient of 100% B to 0% B over 10 minutes.

The second step was a purification of a completely deprotected, i.e.following the removal of the 5'-DMT group, ribozyme by a treatment with2% trifluoroacetic acid or 80% acetic acid on a C4 300 Å 5 μm DeltaPakcolumn in an acetonitrile gradient. Solvents used for this second stepwere: A (0.1M Triethylammonium acetate, pH 6.8) and B (80% acetonitrile,0.1M triethylammonium acetate, pH 6.8). The elution profile was: 5% Bfor 5 minutes, a linear gradient of 5% B to 15% B over 60 minutes, 15% Bfor 10 minutes, and a linear gradient of 15% B to 0% B over 10 minutes.

The fraction containing ribozyme, which is in the triethylammonium saltform, was cooled and lyophilized on a SpeedVac. Solid residue wasdissolved in a minimal amount of ethanol and ribozyme in sodium saltform was precipitated by addition of sodium perchlorate in acetone, (K⁺or Mg²⁺ salts can be produced in an equivalent manner.) The ribozyme wascollected by centrifugation, washed three times with acetone, andlyophilized.

Example 5 HPLC Purification, Anion Exchange column

For a small scale synthesis, the crude material was diluted to 5 mL withdiethylpyrocarbonate treated water. The sample was injected onto eithera Pharmacia Mono Q® 16/10 or Dionex NucleoPac® column with 100% buffer A(10 mM NaClO₄). A gradient from 180-210 mM NaClO₄ at a rate of 0.85mM/void volume for a Pharmacia Mono Q® anion-exchange column or 100-150mM NaClO₄ at a rate of 1.7 mM/void volume for a Dionex NucleoPac®anion-exchange column was used to elute the RNA. Fractions were analyzedby a HP-1090 HPLC with a Dionex NucleoPac® column. Fractions containingfull length product at ≧80% by peak area were pooled.

For a trityl-off large scale synthesis, the crude material was desaltedby applying the solution that resulted from quenching of thedesilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose®Fast Flow column. The column was thoroughly washed with 10 mM sodiumperchlorate buffer. The oligonucleotide was eluted from the column with300 mM sodium perchlorate. The eluent was quantitated and an analyticalHPLC was run to determine the percent full length material in thesynthesis. The eluent was diluted four fold in sterile H₂ O to lower thesalt concentration and applied to a Pharmacia Mono Q® 16/10 column. Agradient from 10-185 mM sodium perchlorate was run over 4 column.volumes to elute shorter sequences, the full length product was theneluted in a gradient from 185-214 mM sodium perchlorate in 30 columnvolumes. The fractions of interest were analyzed on a HP-1090 HPLC witha Dionex NucleoPac® column. Fractions containing over 85% full lengthmaterial were pooled. The pool was applied to a Pharmacia RPC® columnfor desalting.

For a trityl-on large scale synthesis, the crude material was desaltedby applying the solution that resulted from quenching of thedesilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose®Fast Flow column. The column was thoroughly washed with 20 mM NH₄ CO₃H/10% CH₃ CN buffer. The oligonucleotide was eluted from the column with1.5M NH₄ CO₃ H/10% acetonitrile. The eluent was quantitated and ananalytical HPLC was run to determine the percent full length materialpresent in the synthesis. The oligonucleotide was then applied to aPharmacia Resource RPC column. A gradient from 20-55% B (20 mM NH₄ CO₃H/25% CH₃ CN, buffer A=20 mM NH₄ CO₃ H/10% CH₃ CN) was run over 35column volumes. The fractions of interest were analyzed on a HP-1090HPLC with a Dionex NucleoPac® column. Fractions containing over 60% fulllength material were pooled. The pooled fractions were then submitted tomanual detritylation with 80% acetic acid, dried down immediately,resuspended in sterile H₂ O, dried down and resuspended in H₂ O again.This material was analyzed on a HP 1090-HPLC with a Dionex NucleoPac®column. The material was purified by anion exchange chromatography as inthe trityl-off scheme (vide supra).

Example 6 Ribozyme Activity Assay

Purified 5'-end labeled RNA substrates (15-25-mers) and purified 5'-endlabeled ribozymes (˜36-mers) were both heated to 95° C., quenched on iceand equilibrated at 37° C., separately. Ribozyme stock solutions were 1μM, 200 nM, 40 nM or 8 nM and the final substrate RNA concentrationswere ˜1 nM. Total reaction volumes were 50 μL. The assay buffer was 50mM Tris-Cl, pH 7.5 and 10 mM MgCl₂. Reactions were initiated by mixingsubstrate and ribozyme solutions at t=0. Aliquots of 5 μL were removedat time points of 1, 5, 15, 30, 60 and 120 m. Each aliquot was quenchedin formamide loading buffer and loaded onto a 15% denaturingpolyacrylamide gel for analysis. Quantitative analyses were performedusing a phosphorimager (Molecular Dynamics).

Example 7 One pot deprotection of RNA

Applicant has shown that aqueous methyl amine is an efficient reagent todeprotect bases in an RNA molecule. However, in a time consuming step(2-24 hrs), the RNA sample needs to be dried completely prior to thedeprotection of the sugar 2'-hydroxyl groups. Additionally, deprotectionof RNA synthesized on a large scale (e.g., 100 μmol) becomes challengingsince the volume of solid support used is quite large. In an attempt tominimize the time required for deprotection and to simplify the processof deprotection of RNA synthesized on a large scale, applicant describesa one pot deprotection protocol (FIG. 12). According to this protocol,anhydrous methylamine is used in place of aqueous methyl amine. Basedeprotection is carried out at 65° C. for 15 min and the reaction isallowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is thencarried out in the same container for 90 min in a TEA.3HF reagent. Thereaction is quenched with 16 mM TEAB solution.

Referring to FIG. 13, hammerhead ribozyme targeted to site A issynthesized using RNA phosphoramadite chemistry and deprotected usingeither a two pot or a one pot protocol. Profiles of these ribozymes onan HPLC column are compared. The figure shows that RNAs deprotected byeither the one pot or the two pot protocols yield similar full-lengthproduct profiles. Applicant has shown that using a one pot deprotectionprotocol, time required for RNA deprotection can be reduced considerablywithout compromising the quality or the yield of full length RNA.

Referring to FIG. 14, hammerhead ribozymes targeted to site A (from FIG.13) are tested for their ability to cleave RNA. As shown in the FIG. 14,ribozymes that are deprotected using one pot protocol have catalyticactivity comparable to ribozymes that are deprotected using a two potprotocol.

Example 8 Improved protocol for the synthesis of phosphorothioatecontaining RNA and ribozymes using 5-S-Alkyltetrazoles as ActivatingAgent

The two sulfurizing reagents that have been used to synthesizeribophosphorothioates are tetraethylthiuram disulfide (TETD; Vu andHirschbein, 1991 Tetrahedron Letter 31, 3005), and3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent; Vu andHirschbein, 1991 supra). TETD requires long sulfurization times (600seconds for DNA and 3600 seconds for RNA). It has recently been shownthat for sulfurization of DNA oligonucleotides, Beaucage reagent is moreefficient than TETD (Wyrzykiewicz and Ravikumar, 1994 Bioorganic Med.Chem. 4, 1519). Beaucage reagent has also been used to synthesizephosphorothioate oligonucleotides containing 2'-deoxy-2'-fluoromodifications wherein the wait time is 10 min (Kawasaki et al., 1992 J.Med. Chem).

The method of synthesis used follows the procedure for RNA synthesis asdescribed herein and makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5'-end, andphosphoramidites at the 3'-end. The sulfurization step for RNA describedin the literature is a 8 second delivery and 10 min wait steps (Beaucageand Iyer, 1991 Tetrahedron 49, 6123). These conditions produced about95% sulfurization as measured by HPLC analysis (Morvan et al., 1990Tetrahedron Letter 31, 7149). This 5% contaminating oxidation couldarise from the presence of oxygen dissolved in solvents and/or slowrelease of traces of iodine adsorbed on the inner surface of deliverylines during previous synthesis.

A major improvement is the use of an activating agent,5-S-ethyltetrazole or 5-S-methyltetrazole at a concentration of 0.25Mfor 5 min. Additionally, for those linkages which are phosporothioate,the iodine solution is replaced with a 0.05M solution of3H-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage reagent) inacetonitrile. The delivery time for the sulfurization step is reduced to5 seconds and the wait time is reduced to 300 seconds.

RNA synthesis is conducted on a 394 (ABI) synthesizer using a modified2.5 μmol scale protocol with a reduced 5 min coupling step foralkylsilyl protected RNA and 2.5 min coupling step for 2'-O-methylatedRNA. A 6.5-fold excess (162.5 μL of 0.1M=32.5 μmol) of phosphoramiditeand a 40-fold excess of S-ethyl tetrazole (400 μL of 0.25M=100 μmol)relative to polymer-bound 5'-hydroxyl was used in each coupling cycle.Average coupling yields on the 394 synthesizer, determined bycolorimetric quantitation of the trityl fractions, was 97.5-99%. Otheroligonucleotide synthesis reagents for the 394 synthesizer:detritylation solution was 2% TCA in methylene chloride; capping wasperformed with 16% N-Methyl imidazole in THF and 10% aceticanhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM I₂, 49mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile wasused directly from the reagent bottle. S-Ethyl tetrazole solution (0.25Min acetonitrile) was made up from the solid obtained from AppliedBiosystems. Sulfurizing reagent was obtained from Glen Research.

Average sulfurization efficiency (ASE) is determined using the formula:ASE=(PS/Total)^(1/n-1)

where,

PS=integrated ³¹ P NMR values of the P=S diester

Total=integration value of all peaks

n=length of oligo

Referring to tables V and VI, effects of varying the delivery and thewait time for sulfurization with Beaucage's reagent is described. Thesedata suggest that 5 second wait time and 300 second delivery time is thecondition under which ASE is maximum.

Using the above conditions a 36 mer hammerhead ribozyme is synthesizedwhich is targeted to site A. The ribozyme is synthesized to containphosphorothioate linkages at four positions towards the 5' end. RNAcleavage activity of this ribozyme is shown in FIG. 16. Activity of thephosphorothioate ribozyme is comparable to the activity of a ribozymelacking any phosphorothioate linkages.

Example 9 Protocol for the synthesis of 2'-N-phtalimido-nucleosidephosphoramidite

The 2'-amino group of a 2'-deoxy-2'-amino nucleoside is normallyprotected with N-(9-flourenylmethoxycarbonyl) (Fmoc; Imazawa andEckstein, 1979 supra: Pieken et al., 1991 Science 253, 314). Thisprotecting group is not stable in CH₃ CN solution or even in dry formduring prolonged storage at -20° C. These problems need to be overcomein order to achieve large scale synthesis of RNA.

Applicant describes the use of alternative protecting groups for the2'-amino group of 2'-deoxy-2'-amino nucleoside. Referring to FIG. 17,phosphoramidite 17 was synthesized starting from2'-deoxy-2'-aminonucleoside (12) using transient protection withMarkevich reagent (Markiewicz J. Chem. Res. 1979, S, 24). Anintermediate 13 was obtained in 50% yield, however subsequentintroduction of N-phtaloyl (Pht) group by Nefken's method (Nefkens, 1960Nature 185, 306), desilylation (15), dimethoxytrytilation (16) andphosphitylation led to phosphoramidite 17. Since overall yield of thismulti-step procedure was low (20%) applicant investigated somealternative approaches, concentrating on selective introduction ofN-phtaloyl group without acylation of 5' and 3' hydroxyls.

When 2'-deoxy-2'-amino-nucleoside was reacted with 1.05 equivalents ofNefkens reagent in DMF overnight with subsequent treatment with Et₃ N (1hour) only 10-15% of N and 5'(3')-bis-phtaloyl derivatives were formedwith the major component being N-Pht-derivative 15. The N,O-bisby-products could be selectively and quantitively converted to N-Phtderivative 15 by treatment of, crude reaction mixture with cat.KCN/MeOH.

A convenient "one-pot" procedure for the synthesis of key intermediate16 involves selective N-phthaloylation with subsequentdimethoxytrytilation by DMTCl/Et₃ N and resulting in the preparation ofDMT derivative 16 in 85% overall yield as follows. Standardphosphytilation of 16 produced phosphoramidite 17 in 87% yield. One gramof 2'-amino nucleoside, for example 2'-amino uridine (U.S. Biochemicals®part # 77140) was co-evaporated twice from dry dimethyl formamide (Dmf)and dried in vacuo overnight. 50 mls of Aldrich sure-seal Dmf was addedto the dry 2'-amino uridine via syringe and the mixture was stirred for10 minutes to produce a clear solution. 1.0 grams (1.05 eq.) ofN-carbethoxyphthalimide (Nefken's reagent, 98% Jannsen Chimica) wasadded and the solution was stirred overnight. Thin layer chromatography(TLC) showed 90% conversion to a faster moving products (10% ETOH inCHCl₃) and 57 μl of TEA (0.1eq.) was added to effect closure of thephthalimide ring. After 1 hour an additional 855 μl (1.5 eq.) of TEA wasadded followed by the addition of 1.53 grams (1.1 eq.) of DMT-Cl(Lancaster Synthesis®, 98%). The reaction mixture was left to stirovernight and quenched with ETOH after TLC showed. greater than 90%desired product. Dmf was removed under vacuum and the mixture was washedwith sodium bicarbonate solution (5% aq., 500 mls) and extracted withethyl acetate (2×200 mls). A 25 mm×300 mm flash column (75 grams Merckflash silica) was used for purification. Compound eluted at 80 to 85%ethyl acetate in hexanes (yield: 80% purity: >95% by ¹ HNMR).Phosphoramidites were then prepared using standard protocols describedabove.

With phosphoramidite 17 in hand applicant synthesized several ribozymeswith 2'-deoxy-2'-amino modifications. Analysis of the synthesisdemonstrated coupling efficiency in 97-98% range. RNA cleavage activityof ribozymes containing 2'-deoxy-2'-amino-U modifications at U4 and/orU7 positions (see FIG. 1), wherein the 2'-amino positions were eitherprotected with Fmoc or Pht, was identical. Additionally, completedeprotection of 2'-deoxy-2'-amino-Uridine was confirmed bybase-composition analysis. The coupling efficiency of phosphoramidite 17was not effected over prolonged storage (1-2 months) at lowtemperatures.

Other embodiments are within the following claims.

                  TABLE I                                                         ______________________________________                                        Characteristics of Ribozymes                                                  ______________________________________                                        Group I Introns                                                               Size: ˜200 to >1OOO nucleotides.                                        Requires a U in the target sequence immediately 5' of the cleavage            site.                                                                         Binds 4-6 nucleotides at 5' side of cleavage site.                            Over 75 known members of this class. Found in Tetrahymena                     thermophila rRNA, fungal mitochondria, chloroplasts, phage T4,                blue-green algae, and others.                                                 RNAseP RNA (M1 RNA)                                                           Size: ˜290 to 400 nucleotides.                                          RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA                       precursors to form mature tRNA.                                               Roughly 10 known members of this group all are bacterial in origin.           Hammerhead Ribozyme                                                           Size: ˜13 to 40 nucleotides.                                            Requires the target sequence UH immediately 5' of the cleavage                site.                                                                         Binds a variable number nucleotides on both sides of the cleavage             site.                                                                         14 known members of this class. Found in a number of plant                    pathogens (virusoids) that use RNA as the infectious agent (Figure            1)                                                                            Hairpin Ribozyme                                                              Size: ˜50 nucleotides.                                                  Requires the target sequence GUC immediately 3' of the cleavage               site.                                                                         Binds 4-6 nucleotides at 5' side of the cleavage site and a variable          number to the 3' side of the cleavage site.                                   Only 3 known member of this class. Found in three plant pathogen              (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus            and chicory yellow mottle virus) which uses RNA as the infectious             agent.                                                                        Hepatitis Delta Virus (HDV) Ribozyme                                          Size: 50-60 nucleotides (at present).                                         Cleavage of target RNAs recently demonstrated.                                Sequence requirements not fully determined.                                   Binding sites and structurai requirements not fully determined,               although no sequences 5' of cleavage site are required.                       Only 1 known member of this class. Found in human HDV.                        Neurospora VS RNA Ribozyme                                                    Size: ˜144 nucleotides (at present)                                     Cleavage of target RNAs recently demonstrated.                                Sequence requirements not fully determined.                                   Binding sites and structural requirements not fully determined. Only          1 known member of this class. Found in Neurospara VS RNA.                     ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Large-Scale Synthesis                                                                Activator Amidite             % Full                                             Added/Final!                                                                              Added/Final!     Length                                 Sequence (min)       (min)       Time* Product                                ______________________________________                                        A.sub.9 T                                                                              T  0.50/0.33!                                                                              0.1/0.02!  15 m  85                                     A.sub.9 T                                                                              S  0.25/0.17!                                                                              0.1/0.02!  15 m  89                                     (GGU).sub.3 GGT                                                                        T  0.50/0.33!                                                                              0.1/0.02!  15 m  78                                     (GGU).sub.3 GGT                                                                        S  0.25/0.17!                                                                              0.1/0.02!  15 m  81                                     C.sub.9 T                                                                              T  0.50/0.33!                                                                              0.1/0.02!  15 m  90                                     C.sub.9 T                                                                              S  0.25/0.17!                                                                              0.1/0.02!  15 m  97                                     U.sub.9 T                                                                              T  0.50/0.33!                                                                              0.1/0.02!  15 m  80                                     U.sub.9 T                                                                              S  0.25/0.17!                                                                              0.1/0.02!  15 m  85                                     A (36-mer)                                                                             T  0.50/0.33!                                                                              0.1/0.02!  15/15 m                                                                             21                                     A (36-mer)                                                                             S  0.25/0.17!                                                                              0.1/0.02!  15/15 m                                                                             25                                     A (36-mer)                                                                             S  0.50/0.24!                                                                              0.1/0.03!  15/15 m                                                                             25                                     A (36-mer)                                                                             S  0.50/0.18!                                                                              0.1/0.05!  15/15 m                                                                             38                                     A (36-mer)                                                                             S  0.50/0.18!                                                                              0.1/0.05!  10/5 m                                                                              42                                     ______________________________________                                         *Where two coupling times are indicated the first refers to RNA coupling      and the second to 2'-Omethyl coupling. S = 5S-Ethyltetrazole, T =             tetrazole activator. A is 5' -ucu ccA UCU GAU GAG GCC GAA AGG CCG AAA Auc     ccu 3' where lowercase represents 2' -Omethylnucleotides.                

                  TABLE III                                                       ______________________________________                                        Base Deprotection.                                                                                    % Full                                                           Deprotection                                                                             Time          Length                                    Sequence   Reagent    (min)    T °C.                                                                       Product                                   ______________________________________                                        iBu(GGU).sub.4                                                                           NH.sub.4 OH/EtOH                                                                         16 h     55   62.5                                                 MA         10 m     65   62.7                                                 AMA        10 m     65   74.8                                                 MA         10 m     55   75.0                                                 AMA        10 m     55   77.2                                      iPrP(GGU).sub.4                                                                          NH.sub.4 OH/EtOH                                                                         4 h      65   44.8                                                 MA         10 m     65   65.9                                                 AMA        10 m     65   59.8                                                 MA         10 m     55   61.3                                                 AMA        10 m     55   60.1                                      C.sub.9 U  NH.sub.4 OH/EtOH                                                                         4 h      65   75.2                                                 MA         10 m     65   79.1                                                 AMA        10 m     65   77.1                                                 MA         10 m     55   79.8                                                 AMA        10 m     55   75.5                                      A (36-mer) NH.sub.4 OH/EtOH                                                                         4 h      65   22.7                                                 MA         10 m     65   28.9                                      ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        2'-O-Alkylsilyl Deprotection                                                                          % Full                                                            Deprotection                                                                            Time          Length                                    Sequence    Reagent   (min)    T °C.                                                                       Product                                   ______________________________________                                        A.sub.9 T   TBAF      24 h     20   84.5                                                  1.4 M HF  0.5 h    65   81.0                                      (GGU).sub.4 TBAF      24 h     20   60.9                                                  1.4 M HF  0.5 h    65   67.8                                      C.sub.10    TBAF      24 h     20   86.2                                                  1.4 M HF  0.5 h    65   86.1                                      U.sub.10    TBAF      24 h     20   84.8                                                  1.4 M HF  0.5 h    65   84.5                                      B (36-mer)  TBAF      24 h     20   25.2                                                  1.4 M HF  1.5 h    65   30.6                                      A (36-mer)  TBAF      24 h     20   29.7                                                  1.4 M HF  1.5 h    65   30.4                                      ______________________________________                                         B is 5'- UCU CCA UCU GAU GAG GCC GAA AGG CCG AAA AUC CCU                 

                  TABLE V                                                         ______________________________________                                        NMR Data for UC Dimers containing                                             Phosphorothioate Linkage                                                      Synthesis                                                                     #      Type     Delivery Eq.    Wait   ASE (%)                                ______________________________________                                        3524   ribo     2 × 3s                                                                           10.4   2 × 100s                                                                       95.9                                   3525   ribo     2 × 3s                                                                           10.4   2 × 75s                                                                        92.6                                   3530   ribo     2 × 3s                                                                           10.4   2 × 75s                                                                        92.1                                   3526   ribo     1 × 5s                                                                           08.6   1 × 300s                                                                       100.0                                  3578   ribo     1 × 5s                                                                           08.6   1 × 250s                                                                       100.0                                  3529   ribo     1 × 5s                                                                           08.6   1 × 150s                                                                       73.7                                   ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        NMR Data for 15-mer RNA containing                                            Phosphorothioate Linkages                                                     Synthesis                                                                     #       Type     Delivery Eq.   Wait   ASE (%)                                ______________________________________                                        3581    ribo     1 × 5s                                                                           08.6  1 × 250s                                                                       99.6                                   3663    ribo     2 × 4s                                                                           13.8  2 × 300s                                                                       100.0                                  3582    2'-O--Me 1 × 5s                                                                           08.6  1 × 250s                                                                       99.7                                   3668    2'-O--Me 2 × 4s                                                                           13.8  2 × 300s                                                                       99.8                                   3682    2'-O--Me 1 × 5s                                                                           08.6  1 × 300s                                                                       99.8                                   ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "H"stands for C, A or U.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      NNNNUHNNNNN                                                                   (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      NNNNNCUGANGAGNNNNNNCGAAANNNN                                                  (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     The letter "Y"stands for U or C.                                              The letter "H"stands for A, U or C.                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      NNNNNNNYNGHYNNN                                                               (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The letter "N"stands for any base.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      NNNNGAAGNNNNNNNNNNNAAAHANNNNNNNACAUUACNNNNNNNNN                               (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCG60                UCCCCUCGGUAAUGGCGAAUGGGAC85                                                   (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 176 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GGGAAAGCUUGCGAAGGGCGUCGUCGCCCCGAGCGGUAGUAAGCAGGGAACUCACCUCCA60                AUUUCAGUACUGAAAUUGUCGUAGCAGUUGACUACUGUUAUGUGAUUGGUAGAGGCUAAG120               UGACGGUAUUGGCGUAAGUCAGUAUUGCAGCACAGCACAAGCCCGCUUGCGAGAAU176                   __________________________________________________________________________

We claim:
 1. Method for one pot deprotection of RNA comprising,contacting a protected base with anhydrous methyl amine at between 60°C.-70° C. for at least 5 min, cooling the resulting mixture andcontacting said mixture with TEA-3HF reagents under conditions whichremove a protecting group of the 2'-hydroxyl position.
 2. Method ofclaim 1 wherein said RNA is enzymatically active.
 3. Method for one potdeprotection of RNA comprising, contacting a protected base withanhydrous ethyl amine at between 60° C.-70° C. for at least 5 min,cooling the resulting mixture and contacting said mixture with TEA-3HFreagents under conditions which remove a protecting group of the2'-hydroxyl position.